Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3.

Receptor tyrosine kinase regulation of phospholipase C-epsilon (PLC-epsilon), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-gamma1 and PLC-epsilon, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-epsilon, and Rap2B-dependent translocation of PLC-epsilon to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-gamma1 but not of PLC-epsilon. Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-gamma1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-epsilon.

Inhibition of phospholipase C-epsilon by Gi-coupled receptors.

We recently reported that several Gs-coupled receptors stimulate phospholipase C (PLC)-epsilon via increased formation of cyclic AMP and subsequent activation of the small GTPase Rap2B by the cyclic AMP-activated exchange factor Epac1. Here we show by studies in HEK-293 and N1E-115 neuroblastoma cells that this stimulation induced by Gs-coupled receptors or the direct adenylyl cyclase activator, forskolin, is potently inhibited by Gi-coupled receptors, known to inhibit cyclic AMP formation. PLC inhibition by the overexpressed M2 muscarinic receptor and the endogenously expressed sphingosine-1-phosphate and delta-opioid receptors was fully pertussis toxin-sensitive and accompanied by a reduction in Rap2B activation induced by Gs-coupled receptors. In contrast, Rap2B activation and PLC stimulation induced by membrane-permeable cyclic AMP analogues, including an Epac-specific activator, or PLC stimulation caused by constitutively active Rap2B were not affected by the Gi-coupled receptors. In summary, our data indicate that Gi-coupled receptors can inhibit PLC-epsilon, most likely by suppressing formation of cyclic AMP required for Epac-mediated Rap2B activation.

Epac- and Ca2+ -controlled activation of Ras and extracellular signal-regulated kinases by Gs-coupled receptors.

We have recently reported that two typical Gs-coupled receptors, the beta2-adrenergic receptor and the receptor for prostaglandin E1, stimulate phospholipase C-epsilon (PLC-epsilon) and increase intracellular Ca2+ concentration ([Ca2+]i) in HEK-293 cells and N1E-115 neuroblastoma cells, respectively, by a pathway involving Epac1, a cAMP-activated and Rap-specific guanine nucleotide exchange factor (GEF), and the GTPase Rap2B. Here we have demonstrated that these Gs-coupled receptors use this pathway to activate H-Ras and the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Specifically, agonist activation of the receptors resulted in activation of H-Ras and ERK1/2. The latter action was suppressed by dominant negative H-Ras, but not Rap1A. The receptor actions were independent of protein kinase A but fully mimicked by an Epac-specific cAMP analog as well as by a constitutively active Rap2B mutant. On the other hand, a cAMP-binding-deficient Epac1 mutant, the Rap GTPase-activating proteinII, and a dominant negative Rap2B mutant suppressed receptor- and Epac-mediated activation of H-Ras and ERK1/2. Finally, we have demonstrated that activation of H-Ras and ERK1/2 requires the lipase activity of PLC-epsilon and the subsequent [Ca2+]i increase, suggesting that H-Ras activation is mediated by a Ca2+ -activated GEF. In line with this hypothesis, receptor-mediated activation of H-Ras and ERK1/2 was strongly enhanced by expression of RasGRP1, a Ca2+ -regulated Ras-GEF. Collectively, our data indicated that Gs-coupled receptors can activate H-Ras and subsequently the mitogen-activated protein kinases ERK1/2 by a Ca2+ -activated Ras-GEF, possibly RasGRP1, mediated by cAMP-activated Epac proteins, which then lead via Rap2B and PLC-epsilon stimulation to [Ca2+]i increase.